Thermolysin and Bacillus subtilis neutral protease. Conformation and stability of two homologous neutral metalloendopeptidases.
A comparative study on thermolysin from Bacillus thermoproteolyticus and neutral protease from Bacillus subtilis involving (far- and near-ultraviolet circular dichroism (CD) and immunological techniques is reported. These enzymes are homologous metalloendopeptidases, having similar size, kinetic behaviour, substrate specificity and susceptibility to inhibitors. The far-ultraviolet CD spectrum of each protein shows a minimum at 208 and a shoulder near 220 nm; differences in the extent of ellipticity, however, have been observed. Estimates of secondary structure obtained by quantitation of the far-ultraviolet CD spectra indicated a higher helicity of neutral protease relative to thermolysin. In the presence of ethylenediaminetetraacetic acid, which removes calcium and the functional zinc ion from the metalloenzymes, neutral protease is immediately denatured, whereas thermolysin maintains a globular structure, although thermolabile. On the other hand, the zinc-specific chelating agent tetraethylenepentamine does not have measurable effects on the conformation and conformational stability of either protein. Marked higher stability to temperature and guanidine hydrochloride were observed for thermolysin as compared with neutral protease, as indicated by monitoring conformational transitions with CD measurements at 220 nm. Antisera prepared in rabbits using thermolysin as immunogen do not cross-react with neutral protease, indicating differences of surface structure between the two proteins. On the basis and limitations of the techniques employed, it is proposed that the two sequentially and functionally homologous metalloendopeptidases may have similar conformations at specific regions (active and binding sites, at least) of the polypeptide chain essential for biological function, while some variability in the structure of other regions may be tolerated.
D000596:Amino Acids / Q000032:analysis; D001412:Bacillus subtilis / Q000201:enzymology*; D002942:Circular Dichroism; D004355:Drug Stability; D010450:Endopeptidases / Q000378:metabolism*; D007106:Immune Sera; D007118:Immunoassay; D005779:Immunodiffusion; D008667:Metalloproteins / Q000378:metabolism; D011487:Protein Conformation; D013696:Temperature; D013820:Thermolysin / Q000378:metabolism*
D000596:Amino Acids; D007106:Immune Sera; D008667:Metalloproteins; D010450:Endopeptidases; D013820:Thermolysin